Glut4-mediated glucose uptake represents the rate-limiting step of insulin-stimulated glucose disposal. Correspondingly, type 2 diabetes is associated with impaired expression and/or translocation of Glut4 to the cell surface. The insulin-responsive pool of Glut4 is localized in intracellular membrane vesicles, or IRVs, that deliver Glut4 to the plasma membrane. Proteomics analyses performed in our and other laboratories have shown that, in addition to Glut4, these vesicles contain three major proteins: sortilin, IRAP, and LRP1. In the previous funding period, we have shown that sortilin represents the ?proactive? component of the IRVs which is responsible for their formation and insulin responsiveness, whereas other IRV proteins including Glut4 and IRAP may be merely ?passive cargo? in this compartment. This is an unexpected result as sortilin is neither a fat- and skeletal muscle-specific protein, like Glut4, nor it is completely co-localized with Glut4, like IRAP. On the contrary, sortilin is expressed in several types of metabolically active cells and is involved in various trafficking pathways. By now, interaction of the relatively short cytoplasmic tail of sortilin with AP-1, AP-2, PACS-1, GGA, retromer, and several other proteins has been reported. However, molecular mechanisms that control orderly interaction of sortilin with all these trafficking proteins are not known. In Specific Aim 1, we will address this question in relation to the role of sortilin in the biogenesis of the IRVs. This is a central question in the field as insulin-stimulated glucose uptake cannot happen in the absence of these vesicles. To this end, we will map specific binding sites of various adaptors in the cytoplasmic tail of sortilin. We will identify adaptor complexes that are specifically required for the formation of the IRVs and explore mechanisms that regulate their binding. In Specific Aim 2, we will determine how the IRVs are linked to the insulin signaling pathway. In particular, we will test the hypothesis that ?self-assembly? of the IRVs from the individual component proteins creates a platform for the final steps of insulin action. We will explore the role of TBC1D4 and other Akt substrates in this process. Last but not least, we will investigate whether and how the mechanisms of IRV formation and insulin responsiveness are affected by insulin resistance in vitro and in vivo.